Substrate Binding Subsites Research Articles

Protein homology modeling, docking, and phylogenetic analyses of an endo-1,4-β-xylanase GH11 of Colletotrichum lindemuthianum

Endo-1,4-β-xylanase (EC 3.2.1.8) is a crucial enzyme that randomly cleaves the β-1,4-glycosidic linkages of the xylan backbone, releasing xylooligomers of different lengths. The three-dimensional structure of the endo-β-1,4-xylanase protein (xyl1) from Colletotrichum lindemuthianum was modeled and docked with various xylan model compounds. Docking analyses revealed significantly higher stability of C. lindemuthianum XYL1 with the xylopentaose oligomer. Residues interacting with the model oligomers at the respective enzyme active sites were found to be in accord with their role in xylan degradation. Nevertheless, docking analyses of xylanases GH11 from Colletotrichum sp. revealed significative differences in structure, integration of the substrate into the active site, and in the glutamate residues of the catalytic site involved in substrate hydrolysis; of these proteins, 36%, 60%, and 4% integrated xylotetraose, xylopentaose, and xylohexaose in the active site, respectively. Since endoxylanases GH11 from Colletotrichum species interact much more efficiently with xylopentaose and xylotetraose, and xylanases GH11 from different fungi do not seem to have the same substrate binding subsites, we propose that they are enzymes with different affinity to xylooligosaccharides. In agreement with this idea, phylogenetic analyses of xylanases from Colletotrichum sp. show four lineages, suggesting diversifying selection. Most likely, the polydiversity or structural polymolecularity of xylan in plant cell walls processed by these organisms play a determinant role in diversifying selection.

A novel GH6 cellobiohydrolase from Paenibacillus curdlanolyticus B-6 and its synergistic action on cellulose degradation.

We recently discovered a novel glycoside hydrolase family 6 (GH6) cellobiohydrolase from Paenibacillus curdlanolyticus B-6 (PcCel6A), which is rarely found in bacteria. This enzyme is a true exo-type cellobiohydrolase which exhibits high substrate specificity on amorphous cellulose and low substrate specificity on crystalline cellulose, while this showed no activity on substitution substrates, carboxymethyl cellulose and xylan, distinct from all other known GH6 cellobiohydrolases. Product profiles, HPLC analysis of the hydrolysis products and a schematic drawing of the substrate-binding subsites catalysing cellooligosaccharides can explain the new mode of action of this enzyme which prefers to hydrolyse cellopentaose. PcCel6A was not inhibited by glucose or cellobiose at concentrations up to 300 and 100mM, respectively. A good synergistic effect for glucose production was found when PcCel6A acted together with processive endoglucanase Cel9R from Clostridium thermocellum and β-glucosidase CglT from Thermoanaerobacter brockii. These properties of PcCel6A make it a suitable candidate for industrial application in the cellulose degradation process.

Structural and binding properties of laminarin revealed by analytical ultracentrifugation and calorimetric analyses

One of the β-1,3-glucans, laminarin, has been widely used as a substrate for enzymes including endo-1,3-β-glucanase. To obtain quantitative information about the molecular interaction between laminarin and endo-1,3-β-glucanase, the structural properties of laminarin should be determined. The results from pioneering work using analytical ultracentrifugation for carbohydrate analysis showed that laminarin from Laminaria digitata predominantly exists as a single-chain species with approximately 5% of triple-helical species. Differential scanning calorimetry experiments did not show a peak assignable to the transition from triple-helix to single-chain, supporting the notion that a large proportion of laminarin is the single-chain species. The interaction of laminarin with an inactive variant of endo-1,3-β-glucanase from Cellulosimicrobium cellulans, E119A, was quantitatively analyzed using isothermal titration calorimetry. The binding was enthalpically driven and the binding affinity was approximately 106 M−1. The results from binding stoichiometric analysis indicated that on average, E119A binds to laminarin in a 2:1 ratio. This seems to be reasonable, because laminarin mainly exists as a monomer, the apparent molecular mass of laminarin is 3.6 kDa, and E119A would have substrate-binding subsites corresponding to 6 glucose units. The analytical ultracentrifugation experiments could detect different complex species of laminarin and endo-1,3-β-glucanase.

Processivity, Substrate Positioning, and Binding: The Role of Polar Residues in a Family 18 Glycoside Hydrolase

The enzymatic degradation of recalcitrant polysaccharides such as cellulose (β-1,4-linked glucose) and chitin (β-1,4-linked N-acetylglucosamine) by glycoside hydrolases (GHs) is of significant biological and economical importance. In nature, depolymerization is primarily accomplished by processive GHs, which remain attached to the substrate between subsequent hydrolytic reactions. Recent computational efforts have suggested that the processive ability of a GH is directly linked to the ligand binding free energy. The contribution of individual aromatic residues in the active site of these enzymes has been extensively studied. In this study, we offer the first experimental evidence confirming correlation of binding free energy and degree of processivity and evidence that polar residues are essential for maintaining processive ability. Exchanging Thr(276) with Ala in substrate binding subsite -2 in the processive ChiA of Serratia marcescens results in a decrease in both the enthalpy (2.6 and 3.8 kcal/mol) and free energy (0.5 and 2.2 kcal/mol) for the binding to the substrate (GlcNAc)6 and the inhibitor allosamidin, respectively, compared to that of the wild type. Moreover, the initial apparent processivity as measured by [(GlcNAc)2]/[GlcNAc] ratios (17.1 ± 0.4) and chitin degradation efficiency (20%) are greatly reduced for ChiA-T276A versus those of the wild type (30.1 ± 1.5 and 75%, respectively). Mutation of Arg(172) to Ala reduces the level of recognition and positioning of the substrate into the active site. Molecular dynamics simulations indicate ChiA-R172A behaves like the wild type, but the dynamics of ChiA-T276A are greatly influenced by mutation, which is reflective of their influence on processivity.

Substrate recognition and catalysis by LytB, a pneumococcal peptidoglycan hydrolase involved in virulence.

Streptococcus pneumoniae is a major cause of life-threatening diseases worldwide. Here we provide an in-depth functional characterization of LytB, the peptidoglycan hydrolase responsible for physical separation of daughter cells. Identified herein as an N-acetylglucosaminidase, LytB is involved also in colonization and invasion of the nasopharynx, biofilm formation and evasion of host immunity as previously demonstrated. We have shown that LytB cleaves the GlcNAc-β-(1,4)-MurNAc glycosidic bond of peptidoglycan building units. The hydrolysis occurs at sites with fully acetylated GlcNAc moieties, with preference for uncross-linked muropeptides. The necessity of GlcN acetylation and the presence of a single acidic moiety (Glu585) essential for catalysis strongly suggest a substrate-assisted mechanism with anchimeric assistance of the acetamido group of GlcNAc moieties. Additionally, modelling of the catalytic region bound to a hexasaccharide tripentapeptide provided insights into substrate-binding subsites and peptidoglycan recognition. Besides, cell-wall digestion products and solubilisation rates might indicate a tight control of LytB activity to prevent unrestrained breakdown of the cell wall. Choline-independent localization at the poles of the cell, mediated by the choline-binding domain, peptidoglycan modification, and choline-mediated (lipo)teichoic-acid attachment contribute to the high selectivity of LytB. Moreover, so far unknown chitin hydrolase and glycosyltransferase activities were detected using GlcNAc oligomers as substrate.

Biochemical characterization of the novel endo-β-mannanase AtMan5-2 from Arabidopsis thaliana

Plant mannanases are enzymes that carry out fundamentally important functions in cell wall metabolism during plant growth and development by digesting manno-polysaccharides. In this work, the Arabidopsis mannanase 5-2 (AtMan5-2) from a previously uncharacterized subclade of glycoside hydrolase family 5 subfamily 7 (GH5_7) has been heterologously produced in Pichia pastoris. Purified recombinant AtMan5-2 is a glycosylated protein with an apparent molecular mass of 50kDa, a pH optimum of 5.5–6.0 and a temperature optimum of 25°C. The enzyme exhibits high substrate affinity and catalytic efficiency on mannan substrates with main chains containing both glucose and mannose units such as konjac glucomannan and spruce galactoglucomannan. Product analysis of manno-oligosaccharide hydrolysis shows that AtMan5-2 requires at least six substrate-binding subsites. No transglycosylation activity for the recombinant enzyme was detected in the present study. Our results demonstrate diversification of catalytic function among members in the Arabidopsis GH5_7 subfamily.

Mirolase, a novel subtilisin-like serine protease from the periodontopathogen Tannerella forsythia.

The genome of Tannerella forsythia, an etiological factor of chronic periodontitis, contains several genes encoding putative proteases. Here, we characterized a subtilisin-like serine protease of T. forsythia referred to as mirolase. Recombinant full-length latent promirolase [85 kDa, without its signal peptide (SP)] processed itself through sequential autoproteolytic cleavages into a mature enzyme of 40 kDa. Mirolase latency was driven by the N-terminal prodomain (NTP). In stark contrast to almost all known subtilases, the cleaved NTP remained non-covalently associated with mirolase, inhibiting its proteolytic, but not amidolytic, activity. Full activity was observed only after the NTP was gradually, and fully, degraded. Both activity and processing was absolutely dependent on calcium ions, which were also essential for enzyme stability. As a consequence, both serine protease inhibitors and calcium ions chelators inhibited mirolase activity. Activity assays using an array of chromogenic substrates revealed that mirolase specificity is driven not only by the substrate-binding subsite S1, but also by other subsites. Taken together, mirolase is a calcium-dependent serine protease of the S8 family with the unique mechanism of activation that may contribute to T. forsythia pathogenicity by degradation of fibrinogen, hemoglobin, and the antimicrobial peptide LL-37.

Oligosaccharide and Substrate Binding in the Starch Debranching Enzyme Barley Limit Dextrinase

Complete hydrolytic degradation of starch requires hydrolysis of both the α-1,4- and α-1,6-glucosidic bonds in amylopectin. Limit dextrinase (LD) is the only endogenous barley enzyme capable of hydrolyzing the α-1,6-glucosidic bond during seed germination, and impaired LD activity inevitably reduces the maltose and glucose yields from starch degradation. Crystal structures of barley LD and active-site mutants with natural substrates, products and substrate analogues were sought to better understand the facets of LD–substrate interactions that confine high activity of LD to branched maltooligosaccharides. For the first time, an intact α-1,6-glucosidically linked substrate spanning the active site of a LD or pullulanase has been trapped and characterized by crystallography. The crystal structure reveals both the branch and main-chain binding sites and is used to suggest a mechanism for nucleophilicity enhancement in the active site. The substrate, product and analogue complexes were further used to outline substrate binding subsites and substrate binding restraints and to suggest a mechanism for avoidance of dual α-1,6- and α-1,4-hydrolytic activity likely to be a biological necessity during starch synthesis.

Tyrosine sulfation of human trypsin steers S2' subsite selectivity towards basic amino acids.

Human cationic and anionic trypsins are sulfated on Tyr154, a residue which helps to shape the prime side substrate-binding subsites. Here, we used phage display technology to assess the significance of tyrosine sulfation for the specificity of human trypsins. The prime side residues P1′–P4′ in the binding loop of bovine pancreatic trypsin inhibitor (BPTI) were fully randomized and tight binding inhibitor phages were selected against non-sulfated and sulfated human cationic trypsin. The selection pattern for the two targets differed mostly at the P2′ position, where variants selected against non-sulfated trypsin contained primarily aliphatic residues (Leu, Ile, Met), while variants selected against sulfated trypsin were enriched also for Arg. BPTI variants carrying Arg, Lys, Ile, Leu or Ala at the P2′ position of the binding loop were purified and equilibrium dissociation constants were determined against non-sulfated and sulfated cationic and anionic human trypsins. BPTI variants harboring apolar residues at P2′ exhibited 3–12-fold lower affinity to sulfated trypsin relative to the non-sulfated enzyme, whereas BPTI variants containing basic residues at P2′ had comparable affinity to both trypsin forms. Taken together, the observations demonstrate that the tyrosyl sulfate in human trypsins interacts with the P2′ position of the substrate-like inhibitor and this modification increases P2′ selectivity towards basic side chains.

Catalytic Mechanism and Mode of Action of the Periplasmic Alginate Epimerase AlgG

Pseudomonas aeruginosa is an opportunistic pathogen that forms chronic biofilm infections in the lungs of cystic fibrosis patients. A major component of the biofilm during these infections is the exopolysaccharide alginate, which is synthesized at the inner membrane as a homopolymer of 1-4-linked β-D-mannuronate. As the polymer passages through the periplasm, 22-44% of the mannuronate residues are converted to α-L-guluronate by the C5-epimerase AlgG to produce a polymer of alternating β-D-mannuronate and α-L-guluronate blocks and stretches of polymannuronate. To understand the molecular basis of alginate epimerization, the structure of Pseudomonas syringae AlgG has been determined at 2.1-Å resolution, and the protein was functionally characterized. The structure reveals that AlgG is a long right-handed parallel β-helix with an elaborate lid structure. Functional analysis of AlgG mutants suggests that His(319) acts as the catalytic base and that Arg(345) neutralizes the acidic group during the epimerase reaction. Water is the likely catalytic acid. Electrostatic surface potential and residue conservation analyses in conjunction with activity and substrate docking studies suggest that a conserved electropositive groove facilitates polymannuronate binding and contains at least nine substrate binding subsites. These subsites likely align the polymer in the correct register for catalysis to occur. The presence of multiple subsites, the electropositive groove, and the non-random distribution of guluronate in the alginate polymer suggest that AlgG is a processive enzyme. Moreover, comparison of AlgG and the extracellular alginate epimerase AlgE4 of Azotobacter vinelandii provides a structural rationale for the differences in their Ca(2+) dependence.

Biochemical and Structural Characterization of SplD Protease from Staphylococcus aureus

Staphylococcus aureus is a dangerous human pathogen. A number of the proteins secreted by this bacterium are implicated in its virulence, but many of the components of its secretome are poorly characterized. Strains of S. aureus can produce up to six homologous extracellular serine proteases grouped in a single spl operon. Although the SplA, SplB, and SplC proteases have been thoroughly characterized, the properties of the other three enzymes have not yet been investigated. Here, we describe the biochemical and structural characteristics of the SplD protease. The active enzyme was produced in an Escherichia coli recombinant system and purified to homogeneity. P1 substrate specificity was determined using a combinatorial library of synthetic peptide substrates showing exclusive preference for threonine, serine, leucine, isoleucine, alanine, and valine. To further determine the specificity of SplD, we used high-throughput synthetic peptide and cell surface protein display methods. The results not only confirmed SplD preference for a P1 residue, but also provided insight into the specificity of individual primed- and non-primed substrate-binding subsites. The analyses revealed a surprisingly narrow specificity of the protease, which recognized five consecutive residues (P4-P3-P2-P1-P1’) with a consensus motif of R-(Y/W)-(P/L)-(T/L/I/V)↓S. To understand the molecular basis of the strict substrate specificity, we crystallized the enzyme in two different conditions, and refined the structures at resolutions of 1.56 Å and 2.1 Å. Molecular modeling and mutagenesis studies allowed us to define a consensus model of substrate binding, and illustrated the molecular mechanism of protease specificity.

Iterative Saturation Mutagenesis of −6 Subsite Residues in Cyclodextrin Glycosyltransferase from Paenibacillus macerans To Improve Maltodextrin Specificity for 2- O - d -Glucopyranosyl- l -Ascorbic Acid Synthesis

2-O-d-Glucopyranosyl-l-ascorbic acid (AA-2G), a stable l-ascorbic acid derivative, is usually synthesized by cyclodextrin glycosyltransferase (CGTase), which contains nine substrate-binding subsites (from +2 to -7). In this study, iterative saturation mutagenesis (ISM) was performed on the -6 subsite residues (Y167, G179, G180, and N193) in the CGTase from Paenibacillus macerans to improve its specificity for maltodextrin, which is a cheap and easily soluble glycosyl donor for AA-2G synthesis. Site saturation mutagenesis of four sites-Y167, G179, G180, and N193-was first performed and revealed that four mutants-Y167S, G179R, N193R, and G180R-produced AA-2G yields higher than those of other mutant and wild-type CGTases. ISM was then conducted with the best positive mutant as a template. Under optimal conditions, mutant Y167S/G179K/N193R/G180R produced the highest AA-2G titer of 2.12 g/liter, which was 84% higher than that (1.15 g/liter) produced by the wild-type CGTase. Kinetics analysis of AA-2G synthesis using mutant CGTases confirmed the enhanced maltodextrin specificity and showed that compared to the wild-type CGTase, the mutants had no cyclization activity but high hydrolysis and disproportionation activities. A possible mechanism for the enhanced substrate specificity was also analyzed through structure modeling of the mutant and wild-type CGTases. These results indicated that the -6 subsite played crucial roles in the substrate binding and catalytic reactions of CGTase and that the obtained CGTase mutants, especially Y167S/G179K/N193R/G180R, are promising starting points for further development through protein engineering.

Structural Insights into the Hydrolysis and Polymorphism of Methotrexate Polyglutamate by Zebrafish γ-Glutamyl Hydrolase

γ-Glutamyl hydrolases (γGH) catalyze the hydrolysis of γ-linked glutamate residues from the polyglutamyl of folates and antifolates, such as methotrexate (MTX), a widely used anticancer drug. We describe the first crystal structures of the endopeptidase-type γGH (zγGH) from zebrafish and the mutant complexes with MTX(Glu)5 and hydrolyzed MTX(Glu)1, revealing the complete set of key residues involved in hydrolysis as well as the substrate-binding subsites (-1 to +2). The side chain of Phe20 and the 6-methylpterin ring of MTX(Glu)5 invoke π-π interactions to promote distinct concerted conformational alterations involving ∼90° rotations in the complexes with the zγGH-C108A and zγGH-H218N mutant proteins. The structural geometries of the MTX(Glu)5 and hydrolyzed MTX(Glu)1 in the mutant complexes differ significantly from those of the previously known MTX(Glu)1, providing polymorphic information. Together with the structural comparison and the activity analysis, these results shed light on the catalytic mechanism and substrate recognition of zγGH and other γ-glutamyl hydrolases.

Mutation of Tyrosine167Histidine at Remote Substrate Binding Subsite −6 in α-Cyclodextrin Glycosyltransferase Enhancing α-Cyclodextrin Specificity by Directed Evolution

α-Cyclodextrin glycosyltransferase (α-CGTase) can convert starch into α-cyclodextrin with various proportions of β-cyclodextrin and/or γ-cyclodextrin in the products. To improve the α-cyclodextrin-forming specificity, directed evolution on the wild-type α-CGTase was performed by constructing mutant library with error-prone PCR method. The positive mutant strains were selected in combination of starch plate screening with HPLC detection of the products. An α-CGTase from the mutant strain (assigned No. 95) was found to be able to increase the α:β ratio in product mixture from 3.4 to 7.8 in comparison with the wild-type α-CGTase. Sequence alignment indicated that two mutations occurred in the No. 95 mutant α-CGTase, which were Y167H and A536V. Reverse mutation revealed that Y167H was responsible for this change. A series of 167 site-substituted mutants could improve the α:β ratio to different extents as indicated by saturated mutagenesis, with Y167H as the best substitution. In conclusion, Y167 was confirmed to be one of the main subsites in the -6 domain of α-CGTase that is responsible for the α:β ratio in the product mixture. Y167H is most preferable among all types of mutant enzymes tested at this site. The reconstructed Y167H (i.e., No. 95) α-CGTase showed better potential for α-cyclodextrin production on industrial scale.

Substrate recognition mechanism of a glycosyltrehalose trehalohydrolase from Sulfolobus solfataricus KM1.

Glycosyltrehalose trehalohydrolase (GTHase) is an α-amylase that cleaves the α-1,4 bond adjacent to the α-1,1 bond of maltooligosyltrehalose to release trehalose. To investigate the catalytic and substrate recognition mechanisms of GTHase, two residues, Asp252 (nucleophile) and Glu283 (general acid/base), located at the catalytic site of GTHase were mutated (Asp252→Ser (D252S), Glu (D252E) and Glu283→Gln (E283Q)), and the activity and structure of the enzyme were investigated. The E283Q, D252E, and D252S mutants showed only 0.04, 0.03, and 0.6% of enzymatic activity against the wild-type, respectively. The crystal structure of the E283Q mutant GTHase in complex with the substrate, maltotriosyltrehalose (G3-Tre), was determined to 2.6-Å resolution. The structure with G3-Tre indicated that GTHase has at least five substrate binding subsites and that Glu283 is the catalytic acid, and Asp252 is the nucleophile that attacks the C1 carbon in the glycosidic linkage of G3-Tre. The complex structure also revealed a scheme for substrate recognition by GTHase. Substrate recognition involves two unique interactions: stacking of Tyr325 with the terminal glucose ring of the trehalose moiety and perpendicularly placement of Trp215 to the pyranose rings at the subsites -1 and +1 glucose.

Mechanistic Insights into the 1,3-Xylanases: Useful Enzymes for Manipulation of Algal Biomass

Xylanases capable of degrading the crystalline microfibrils of 1,3-xylan that reinforce the cell walls of some red and siphonous green algae have not been well studied, yet they could prove to be of great utility in algaculture for the production of food and renewable chemical feedstocks. To gain a better mechanistic understanding of these enzymes, a suite of reagents was synthesized and evaluated as substrates and inhibitors of an endo-1,3-xylanase. With these reagents, a retaining mechanism was confirmed for the xylanase, its catalytic nucleophile identified, and the existence of -3 to +2 substrate-binding subsites demonstrated. Protein crystal X-ray diffraction methods provided a high resolution structure of a trapped covalent glycosyl-enzyme intermediate, indicating that the 1,3-xylanases likely utilize the (1)S(3) → (4)H(3) → (4)C(1) conformational itinerary to effect catalysis.

Enhancement of the Catalytic Activity of a 27 kDa Subtilisin-Like Enzyme from Bacillus amyloliquefaciens CH51 by in Vitro Mutagenesis

AprE51 from Bacillus amyloliquefaciens CH51 is a 27 kDa subtilisin-like protease with fibrinolytic activity. To enhance the catalytic activity of AprE51, two residues, Gly-169 and Ser-101, which, according to the three-dimensional structural model of subtilisin, are located in the P1 substrate-binding site and S3 subsite, respectively, were mutated by site-directed mutagenesis. Results of the mutational analysis showed that substitution of alanine for Gly-169 increased the fibrinolytic activity 1.4-fold. All four Ser-101 mutations, that is, replacements with arginine, leucine, lysine, and tryptophan, also increased the fibrinolytic activity up to 3.9-fold. The S101W mutant with a bulky side chain was more active than mutants with a positively charged or nonpolar small side chains. The fibrinolytic activity of the S101W mutant was further increased by error-prone polymerase chain reaction. The AprE51-6 mutant (S101W/G169A/V192A) had stronger fibrinolytic activity than the S101W mutant. Purified AprE51-6 had a 2.5-fold higher k(cat) and a 2.3-fold lower K(m), which resulted in a 6-fold increase in catalytic efficiency (k(cat)/K(m)) relative to that of wild-type AprE51. In addition, AprE51-6 showed a relatively broader pH range and increased thermostability as compared to AprE51.

Crystal Structure of α-Galactosidase from Lactobacillus acidophilus NCFM: Insight into Tetramer Formation and Substrate Binding

Lactobacillus acidophilus NCFM is a probiotic bacterium known for its beneficial effects on human health. The importance of α-galactosidases (α-Gals) for growth of probiotic organisms on oligosaccharides of the raffinose family present in many foods is increasingly recognized. Here, the crystal structure of α-Gal from L. acidophilus NCFM (LaMel36A) of glycoside hydrolase (GH) family 36 (GH36) is determined by single-wavelength anomalous dispersion. In addition, a 1.58-Å-resolution crystallographic complex with α-d-galactose at substrate binding subsite −1 was determined. LaMel36A has a large N-terminal twisted β-sandwich domain, connected by a long α-helix to the catalytic (β/α)8-barrel domain, and a C-terminal β-sheet domain. Four identical monomers form a tightly packed tetramer where three monomers contribute to the structural integrity of the active site in each monomer. Structural comparison of LaMel36A with the monomeric Thermotoga maritima α-Gal (TmGal36A) reveals that O2 of α-d-galactose in LaMel36A interacts with a backbone nitrogen in a glycine-rich loop of the catalytic domain, whereas the corresponding atom in TmGal36A is from a tryptophan side chain belonging to the N-terminal domain. Thus, two distinctly different structural motifs participate in substrate recognition. The tetrameric LaMel36A furthermore has a much deeper active site than the monomeric TmGal36A, which possibly modulates substrate specificity. Sequence analysis of GH36, inspired by the observed structural differences, results in four distinct subgroups having clearly different active-site sequence motifs. This novel subdivision incorporates functional and architectural features and may aid further biochemical and structural analyses within GH36.

Substrate specificity of Staphylococcus aureus cysteine proteases – Staphopains A, B and C

Human strains of Staphylococcus aureus secrete two papain-like proteases, staphopain A and B. Avian strains produce another homologous enzyme, staphopain C. Animal studies suggest that staphopains B and C contribute to bacterial virulence, in contrast to staphopain A, which seems to have a virulence unrelated function. Here we present a detailed study of substrate preferences of all three proteases. The specificity of staphopain A, B and C substrate-binding subsites was mapped using different synthetic substrate libraries, inhibitor libraries and a protein substrate combinatorial library. The analysis demonstrated that the most efficiently hydrolyzed sites, using Schechter and Berger nomenclature, comprise a P2–Gly↓Ala(Ser) sequence motif, where P2 distinguishes the specificity of staphopain A (Leu) from that of both staphopains B and C (Phe/Tyr). However, we show that at the same time the overall specificity of staphopains is relaxed, insofar as multiple substrates that diverge from the sequences described above are also efficiently hydrolyzed.

C‐Terminal extension of a plant cysteine protease modulates proteolytic activity through a partial inhibitory mechanism

The amino acid sequence of ervatamin-C, a thermostable cysteine protease from a tropical plant, revealed an additional 24-amino-acid extension at its C-terminus (CT). The role of this extension peptide in zymogen activation, catalytic activity, folding and stability of the protease is reported. For this study, we expressed two recombinant forms of the protease in Escherichia coli, one retaining the CT-extension and the other with it truncated. The enzyme with the extension shows autocatalytic zymogen activation at a higher pH of 8.0, whereas deletion of the extension results in a more active form of the enzyme. This CT-extension was not found to be cleaved during autocatalysis or by limited proteolysis by different external proteases. Molecular modeling and simulation studies revealed that the CT-extension blocks some of the substrate-binding unprimed subsites including the specificity-determining subsite (S2) of the enzyme and thereby partially occludes accessibility of the substrates to the active site, which also corroborates the experimental observations. The CT-extension in the model structure shows tight packing with the catalytic domain of the enzyme, mediated by strong hydrophobic and H-bond interactions, thus restricting accessibility of its cleavage sites to the protease itself or to the external proteases. Kinetic stability analyses (T(50) and t(1/2) ) and refolding experiments show similar thermal stability and refolding efficiency for both forms. These data suggest that the CT-extension has an inhibitory role in the proteolytic activity of ervatamin-C but does not have a major role either in stabilizing the enzyme or in its folding mechanism.